Developing Rat Bone Marrow Derived Mast Cells by the Splenic Cells Culture Supernatant of Rat and Mouse.

Background: Mast cells play a critical role in the pathogenesis of various immunological and non-immunological diseases. It is now accepted that culturing primary mast cells considered as a tool for investigation role of mast cells in diseases. Development of various animal primary mast cells and their function could be used for the translational studies in the pathogenesis of human diseases. The aim of the study was to develop simple and cost-efficient method for differentiation and culture of rat mast cells from bone marrow by using rat and mouse spleen supernatant. Materials and Methods: Bone marrow cells from 10 to15-weeks-old male rats was obtained and cultured for three weeks on cell culture medium. After that, purity of cells was approved by FCɛRI and CD117 antibodies, toluidine blue and Immunohistochemistry (IHC). Results: After 3 weeks continuous culturing, high purity of cells was found. CD117, CD34 expression and tryptase were 80.1, 76.89 and 87.9%, respectively by rat splenic supernatant, whereas 85.4, 83.07 and 82.1%, respectively with mouse splenic supernatants. Besides, rat spleen supernatant developed 91.4% and mouse splenocyte supernatant developed 89.7% mast cells based on surface markers. Conclusion: The data presented in this study indicated equal maturation and differentiation of bone marrow derived rat mast cells by using both spleen supernatants.


INTRODUCTION
Mast Cells (MC) are paracrine cells playing important role in allergic and non-allergic reactions (1). These cells contain receptors with high affinity for immunoglobulin E (IgE) (1,2). Due to difficulty in isolating high numbers of mast cells from animals, cell lines are commonly used for biological and functional investigations (3). For example, the biology of mast cells has been limited to a species of mast cells known as connective tissue mast cells, as they could be easily obtained, especially from the peritoneal cavity. Moreover, study of mucosal mast cells and basophils have been neglected due to difficulties in their isolation (4). Emerging data, identified factors controlling the growth and differentiation of human mast cells. Stem Cell Factor (SCF) and IL-3 were considered as a source of stimulator of mast cells in in-vitro many years ago (5).
However, IL-3 does not seem to be a growth and differentiation factor for (normal) human mast cells in bone marrow or peripheral blood cells culture (4). CD117 receptor which is specific for mast cells binds SCF and accounts for maturation and differentiation of mast cells.
IL-3 is the major differentiating factor for basophils and others inflammatory cells (6). Recently, human mast cells growth was successfully induced in long-term cultures of umbilical cord blood cells by a fibroblastic cell line (4).
Mast cells can be generated from different sources for in vitro studies (7). These cells nowadays are considered for the studies of reconstructive investigations (8). Studies performed on mast cells have been done mostly on BALBc mice (9). There is few exceptions in mast cells of rat and mice for example proliferation, quantity of metachromatic granules (10, 11), and life span. Important issue is life span of rat mast cells, which is shorter (5 weeks) than the mice (8-12 weeks) (11, 12). Finding the differences of mast cells of two species is important in mast cell biology investigation. Thus, the current study aimed to characterize the isolation protocols of these cells by using two kinds of stimulations for proliferation and differentiations of cells i.e. spleen supernatant culture from mouse or rat sources. In this way femur isolated bone marrow cells were cultured in two different conditions with rat splenic cells supernatants or mouse splenic cell supernatants origin for 3 weeks. After 3 weeks the purity and specific markers of cells were evaluated by Immunohistochemistry (IHC) and flow cytometry analysis.

Bone Marrow cell isolation
The study was carried out based on the International Association for the Study of Pain (IASP) (13) and research board of Urmia University which approved all the experiments sets. All rats were first anesthetized by intraperitoneal Ketamine-Xylazine (ketamine 5%, 90 mg/kg and xylazine 2%, 5 mg/kg) and then euthanized by a highdose of CO2 (2).
Bone marrow cells were immediately isolated from rat femur and tibia bones as described earlier (14). Then the bones were flushed by insulin syringe using endotoxin-free culture medium and obtained materials were centrifuged for 10 minutes at 320xg at 4C. Then cells were cultured at the ratio of 0.5×10 6 /ml in complete media (RPMI1640 containing FBS 10%, 100 IU/ml Penicillin, 100 µg/ml Streptomycin, 0.1 µmol of non-essential aminoacids, 2 mmol L-glutamine) and splenic mitogen pokeweed (20%).
The medium was changed every 5 days. After 3-4 weeks, cells were washed with cold PBS (1X) and then evaluated as described in next part.

ICC of CD34 and CD117
The cells are placed on a slide and were fixed with paraformaldehyde 4% and then slides were washed with PBS for 10 minutes and entered to the specific staining stage afterward. Immunocytochemistry staining steps were Then the slides were flooded in PBS for 5 minutes between each two steps. Then the cells were counterstained by Gill's II hematoxylin (Fisher Scientific, Fair Lawn, NJ,) and fixed using crystal/mount (Biomeda, Foster City, CA, USA) and prepared for the study (17) .

ICC (Immunocytochemistry) of Tryptase
The cells were placed on a slide and fixed with paraformaldehyde 4%. Then the slides were washed in PBS for 10 minutes and later enter the specific staining stage.
Slides were immersed in sodium citrate buffer to retrieve antigen, pH 6.0, in an 86 °C water bath for 15 min. After being rinsed with water, protocols were followed by incubation in PBS for 5 min, and then slides were blocked and mounted with Crystal/Mount (Biomeda, Foster City, CA) as described earlier (18).

Immunocytochemical analysis of mast cells
In the next step, specific markers for differentiated bone    Compared to other myeloid cells, not much information for the mast cell biology is available, which could be due to be their low number (0.002-0.05%) and difficult in isolation procedures (20). Although sampling of bone marrow cells is a prominent method for obtaining mast cells, its sensitivity and accuracy are limited due to the heterogeneous origin and a low yield of mast cells.  (29). Mast cells are activated through the stimulants such as cross-linking IgE, and SCF (30). Activation and differentiation of mast cells are regulated by IL-3 and SCF, respectively (31). C-kit , CD117, and SCF receptors are important factors for survival, differentiation, and maturation of MCs (29,32). Although SCF is the essential factor for mast cells survival, FCƐRI can also improve their viability (30). In the rat, IL-3 is known as a mast cell growth factor (33), thus mast cells are generally prepared from bone marrow cells using culture with IL-3 or a supernatant containing IL-3 (34). Recent reports have demonstrated that SCF is another c-kit ligand as mast cell growth factor in splenic cells culture (35). Several types of factors have been identified which can affect progenitor hematopoietic cells for the lineage differentiation. A suspension pokeweed with stimulatory mitogens of splenic cells produces medium condition and is able to stimulate ancestral cells of granulocyte-macrophage, eosinophils, megakaryocytes colony, and mast cells generation and differentiation (36). CD4+ T lymphocytes produce a significant amount of IL-3 in spleen supernatants. SCF is produced by fibroblasts in laboratory conditions (37).
These cells exist in the splenic connective tissue and produce SCF and IL-3 in in-vitro conditions for survival and spread of mast cells (7). Thus, it has been concluded that rat mast cells development by mouse spleen supernatants is the same as rat spleen supernatants.
Finally, here, we propose a practical and inexpensive method for purifying rat mast cells and conditions for generating pure cultured rat mast cell from bone marrow.